Iron deficiency (ID) is the most prevalent single deficiency state on a worldwide basis. It is important economically because it diminishes the capability of individuals who are affected to perform physical labor, and it diminishes both growth and learning in children.
Absolute iron deficiency, with anemia or without anemia, and functional iron deficiency (FID) are high frequency clinical conditions, and these patients have iron deficient erythropoiesis. Absolute iron deficiency is defined as a decreased total iron body content. Iron deficiency anemia (IDA) occurs when iron deficiency is sufficiently severe to diminish erythropoiesis and cause the development of anemia. Functional iron deficiency describes a state where the total iron content of the body is normal or even elevated, but the iron is ‘locked away’ and unavailable for the production of red blood cells. This condition is observed mainly in patients with chronic renal failure who are on hemodialysis, and in patients with chronic inflammation or chronic infections.
Iron status can be measured using hematological and biochemical indices. Each parameter of iron status reflects changes in different body iron compartments and is affected at different levels of iron depletion. Specific iron measurements include hemoglobin (Hgb), mean cell volume (MCV), hematocrit (Hct), erythrocyte protoporphyrin, plasma iron, transferrin, transferrin saturation levels (TSAT), serum ferritin (SF) and more recently soluble transferrin receptors (sTfR) and red-cell distribution width (RDW).
Typical values for normal iron status are SF 100±60 ng/ml and Hgb 12-17 g/dl for women and 14-19 g/dl for men. The typical values for iron deficiency anemia are SF <22 ng/ml, Hgb for women <12 g/dl and for men <13 g/dl.
Hemoglobin (Hgb) has been used longer than any other iron status parameter. It provides a quantitative measure of the severity of iron deficiency once anemia has developed. Hemoglobin determination is a convenient and simple screening method and is especially useful when the prevalence of iron deficiency is high, as in pregnancy or infancy. The limitations of using hemoglobin as a measure of iron status are its lack of specificity (as factors such as vitamin B12 or folate deficiency, genetic disorders and chronic infections can limit erythropoiesis) and its relative insensitivity due to the marked overlap in values between normal and iron deficient populations. To identify iron deficiency anemia, hemoglobin is measured together with more selective measurements of iron status.
A reduction in mean cell volume (MCV) occurs when iron deficiency becomes severe, at about the same time as anemia starts to develop. It is a fairly specific indicator of iron deficiency once thalassemia and the anemia of chronic disease have been excluded. A cut-off value of 80 fl is accepted as the lower limit of normal in adults. It has been reported that when measured on Technicon hematology analyzers (that use optical measurement of red blood cells) iron deficiency blood samples have reduced mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC). However, when measured by impedance-based hematology analyzers (Such as Coulter or Sysmex instruments) MCHC is insensitive but more specific for iron deficiency (Bain, B. J., Blood Cells, A Practical Guide, Second Edition, Blackwell Science Ltd., 1995, Chapter 8, pages 197-199). The red-cell distribution width (RDW) has been used recently in combination with other parameters for the classification of anemias. It reflects the variation in the size of the red cells and can be used to detect subtle degrees of anisocytosis.
The most commonly used iron status parameters at present are transferrin saturation (TSAT) and serum ferritin (SF). However, both are indirect measures of iron status. Transferrin is a transport protein that contains two iron binding sites by which it transports iron from storage sites to erythroid precursors. TSAT (i.e., the percentage of total binding sites that are occupied by iron) is a measure of iron that is available for erythropoiesis. TSAT is calculated by dividing the serum iron by the total iron binding capacity (TIBC), a measurement of circulating transferrin, and multiplying by 100. Ferritin is a storage protein that is contained primarily within the reticuloendothelial system, with some amounts released in the serum. Under conditions of iron excess, ferritin production increases to offset the increase in plasma iron. The level of ferritin in the serum, therefore, reflects the amount of iron in storage.
Definition of Functional Iron Deficiency (FID) and AbsoluteIron Deficiency (AID) by Kidney Disease Outcomes, QualityInitiative K/DOQI (U.S.A)Ferritin μg/L<100100–800TSAT <20%AIDTSAT <20%FID
For patients with chronic kidney disease, absolute iron deficiency may be diagnosed when TSAT is <20% and SF is <100 ng/ml. Functional iron deficiency may be more difficult to diagnose since iron status parameters may indicate adequate iron stores. There are different criteria in defining FID, one of them is published by the Kidney Disease Outcomes Quality Initiative—K/DOQI (Eknoyan G, et al. Continuous quality improvement: DOQI becomes K/DOQI and is updated. National Kidney Foundation's Dialysis Outcomes Quality Initiative. Am J Kidney Dis., 2001 January; 37(1):179-194; Anemia Management in Chronic Kidney Disease: Role of Factors Affecting Epoetin Responsiveness, ESCHBACH, J., J Am Soc Nephrol 13: 1412-1414, 2002.), as shown in the table above.
The limitations of using transferrin saturation reflect those of serum iron, i.e., wide diurnal variation and low specificity. TSAT is also reduced in inflammatory disease. Transferrin saturation is commonly used in population studies combined with other indicators of iron status. On the other hand, as ferritin is an acute phase reactant, its serum levels may be elevated in the presence of chronic inflammation, infection, malignancy and liver disease. Alcohol consumption has also been suggested to independently raise serum ferritin.
Recently, several new red blood cell and reticulocyte parameters have been reported having utilities in detection of iron deficiency and functional iron deficiency. Two of the parameters are hypochromic red cell percentage (referred to as % Hypo) and CHr (reticulocyte hemoglobin content) reported by the Bayer ADVIA 120 hematology analyzer (Thomas et al., Biochemical Markers and Hematologic Indices in the Diagnosis of Functional Iron Deficiency, Clinical Chemistry 48:7, 1066-1076, 2002). Hypochromic red cell percentage is defined as the percentage of red blood cells having hemoglobin less than 28 g/dl. CHr is defined by the formula (CHr=MCVr×CHCMr), wherein MCVr is the mean reticulocyte cell volume and CHCMr is the mean hemoglobin concentration of reticulocytes which is obtained by an optical cell-by-cell hemoglobin measurement.
Reticulocytes are immature red blood cells with a life span of only 1 to 2 days. When these are first released from the bone marrow, measurement of their hemoglobin content can provide the amount of iron immediately available for erythropoiesis. A less than normal hemoglobin content in these reticulocytes is an indication of inadequate iron supply relative to demand. The amount of hemoglobin in these reticulocytes also corresponds to the amount of hemoglobin in mature red blood cells. CHr has been evaluated recently in numerous studies as a test for iron deficiency and functional iron deficiency and has been found to be highly sensitive and specific. However, exact threshold values have not been established, as the threshold values vary depending on the laboratory and instrument used.
Epoetin is effective in stimulating production of red blood cells, but without an adequate iron supply to bind to heme, the red blood cells will be hypochromic, i.e., low in hemoglobin content. Thus, in states of iron deficiency, a significant percentage of red blood cells leaving the bone marrow will have a low hemoglobin content. By measuring the percentage of red blood cells with hemoglobin content <28 g/dl, iron deficiency can be detected. % Hypo >10% has been correlated with iron deficiency, and hence has been used as a diagnostic criterion for detection of iron deficiency (Revised European Best Practice Guidelines for the Management of Anaemia in Patients With Chronic Renal Failure, Locatelli, F. et al., Nephrology and Dyalisis Transplantation, Volume 19 May 2004 (Supplement 2), Guideline III.2, page ii22-24).
% Hypo is a reported parameter on several Bayer hematology analyzers based on an optical cell-by-cell hemoglobin measurement. % Hypo must be measured using a fresh whole blood sample (less than four hours after blood collection), since storage or sample aging leads to erroneous increases of % Hypo report due to red blood cell swelling (Revised European Best Practice Guidelines for the Management of Anaemia in Patients With Chronic Renal Failure, Locatelli, F. et al., Nephrology and Dyalisis Transplantation, Volume 19 May 2004 (Supplement 2), Appendix B, page ii39-41).
Two other parameters have been reported recently correlating to % Hypo and CHr are RBC-Y and Ret-He reported by the Sysmex XE-2100 hematology analyzer (Machin S. J. et al. Functional Iron Deficiency and New Red Cell Parameters on the Sysmex XE-2100, ISLH 2001 Industry-Sponsored Workshops, ISLH XIVth International Symposium, 2001; and Thomas, C. et al., Anemia of Chronic Disease: Pathophysiology and Laboratory Diagnosis, Laboratory Hematology 2005, 11:14-23). RBC-Y is the mean value of the forward light scatter histogram within the mature erythrocyte population, and Ret-He is the mean value of the forward light scatter histogram within the reticulocyte population obtained in a reticulocyte measurement on the Sysmex XE-2100 hematology analyzer.
Most recently, several functions of red blood cell parameters as well as reticulocyte parameters have been disclosed by Simon-Lopez in the co-pending application Ser. No. 11/524,682 to be useful in detection of iron deficiency. These include a RBC size function (RSf) defined as a product function of MCV and MRV, a volume-hemoglobin factor (VHf) defined as a product function of MCV and Hgb, a volume-hemoglobin/distribution factor (VHDWf) defined as a function of MCV, Hgb and RDW.
It has been recognized that CHr and % Hypo are only provided on Bayer's hematology analyzers. Therefore, this information is not available for many clinical laboratories and hospitals. On the other hand, MCV, MCH, MCHC and Hgb are the traditional red cell indices provided on all automated hematology analyzers. Because of the availability of these traditional red blood cell parameters, it is desirable to develop diagnostic indicators using these parameters for detection iron deficiency with comparable clinical accuracy, sensitivity and specificity to the known parameters such as CHr and % Hypo.